DESCRIPTION: Glycans have several distinct properties that make them excellent targets for disease biomarkers. Firstly, the location of the glycans on the cell surface makes them the first point of contact of cellular interactions and thus crucial in the control of normal metabolic processes. Cell surface molecules are also strategically exposed for surveillance by the immune system allowing for the potential of immune recognition of abnormal cells. Secondly, specific glycan structures that are not present, or are in low amounts, in normal states proliferate in disease states. And lastly, changes in glycosylation involve many proteins, including those that are highly abundant. Therefore, a single change in a cell's glycosylation machinery can affect many different glycoconjugates. To effectively employ and discover glycan disease markers new glycan-specific reagents are urgently needed. Using computational methods to guide molecular evolution, carbohydrate-processing enzymes will be converted into high affinity receptor proteins that retain the native specificity of the enzyme, but which no longer have enzyme activity. Because such a protein has lectin-like properties, but is derived from an enzyme, we are calling them Lectenz(r). Lectenz(r) have several potential advantages over lectins and antibodies as glycomics reagents, including precise definition of specificity, ease of preparation in a monovalent form, and (for human homologues) minimal in vivo toxicity. In this proposal, the peptide N-glycanase F (PNGase F) carbohydrate-processing enzyme will be converted into a high-specificity affinity reagent for peptides and proteins that contain asparagine-linked carbohydrate chains. The Lectenz(r) based on PNGase F may be employed directly to address the needs of glycomics/proteomics analysis through sample enrichment, thus facilitating glycosylation site-mapping. Glycosylation site mapping is currently extremely tedious to perform and yet is essential in fully characterizing and exploiting glycans as markers of specific disease states. The principle advantages of an engineered Lectenz(r) are that the Lectenz(r) is specific to a defined carbohydrate sequence, and, in contrast to antibodies, will recognize that sequence in a broad range of glycan contexts. Further, in contrast to plant lectins, engineered Lectenz(r) are derived from enzymes that have exquisite substrate specificities and low toxicities. Lastly, as there is an abundance of carbohydrate- processing enzymes known, it is possible to employ the Lectenz(r) technology (patent pending) to assemble panels of affinity reagents tailor-made for characterizing, monitoring or detecting specific glycans.